Structural features of the polypurine tract primer of (+) strand DNA synthesis mediating its recognition by the RNase H domain of HIV-1RT have been examined. Such studies have indicated that template nucleobases can be removed without loss of cleavage specificity at the scissile -1/+1 phosphodiester bond, while equivalent lesions in the RNA primer were inhibitory. Subsequent studies identified individual bases of the PPT primer that are critical to its function. The use of purine analogs has also allowed us to determine the role of the exocyclic groups of the purine ring at these critical positions. Finally, using a combination of analog-substituted substrates and mutants of HIV-1 RT, nucleoside analogs have been used to understand how the single stranded template interacts with residues of the fingers subdomain prior to accessing the DNA polymerase active site of HIV-1 RT.